Search results for: kinase assays

Authors: Lynn Lehmann, Dinorah Leyva, Ana Lazic, Stefan Duhr, Philipp Baaske

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Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes and is essential for the development of drug diagnostics and therapeutics. Here we present a novel, label-free, and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by Microscale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates and blood serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. We demonstrate how MST determines the picomolar affinities of antibody::antigen interactions, and protein::protein interactions measured from directly from cell lysates. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Keywords: biochemistry, biophysics, molecular interactions, quantitative techniques

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Authors: Mulanga Luscious Mulaudzi, Ignatious Ncube

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In nature, enzymatic degradation of lignocellulose is more efficient compared to in vivo bioprocessing. Thus, a co-culture should enable production of more efficient enzyme preparations that would mimic the natural decomposition of lignocellulose. The aim of the study was to establish a microbial co-culture for the production of highly active cellulase preparations. The objectives were to determine the use of a variety of culture media to isolate cellulose degrading microorganisms from decomposing banana pseudo stem and to optimize production of cellulase by co-cultures of microorganisms producing high levels of cellulose. Screening of fungal isolates was done on carboxylmethylcellulose agar plates which were stained with Congo red to show hydrolytic activity of the isolates. Co-culture and mixed culture of these microorganisms were cultured using Mendel salts with Avicel as the carbon source. Cultures were incubated at 30 °C with shaking at 200 rpm for 240 hrs. Enzyme activity assays were performed to determine endoglycosidase and β-glucosidase. Mixed culture of fungi-dead bacterial cells showed to be the best co-culture/ mixed culture to produce higher levels of cellulase activity in submerged fermentations (SmF) using Avicel™ as a carbon source. The study concludes use microorganism 5A in co-cultures is highly recommended in order to produce high amounts of β-glucosidases, no matter the combination used.

Keywords: avicel, co-culture, submerged fermentation, pseudostem

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Authors: Paras Agarwal

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Background: Initially believed to be rare, metastases to the eye are the most common ocular malignancy. The choroid’s high perfusion rate not only makes it the most susceptible ocular site for tumour seeding, but also promotes its growth. The cancers most frequently responsible for choroidal metastases originate from the breast and lung, although a significant proportion have unidentified primaries at the time of presentation. Case Presentation: This case report describes a 34 year old female presenting to the ophthalmology department with a one month history of painless distorted vision. On fundus examination, she was noted to have bilateral choroidal lesionsand subsequently underwent a comprehensive diagnostic work-up. The patient was diagnosed with metastatic pulmonary adenocarcinoma, despite lacking conventional risk factors. As she was found to have a mutation in EGFR, the patient was commenced on tyrosine-kinase inhibition with afatinib. The choroidal lesions regressed with a significant improvement in visual acuity and a dramatic anatomical reduction of the choroidal masses. Conclusions: Our case demonstrates the importance of considering metastases as a differential diagnosis for choroidal lesions. Appropriate and thorough history-taking, examination and investigations may be required in order to deduce the underlying cause. Our case is unusual in view of the choroidal lesion being the primary manifestation of metastatic lung cancer in a young patient with no known risk factors. Early recognition of choroidal metastases is important as it is often the first sign of tumour dissemination and will prompt earlier treatment with systemic medications such as chemotherapy, immunotherapy, targeted therapy or hormonal therapy. Our case report also demonstrates the efficacy of afatinib for the treatment of choroidal metastases, with morphological and functional improvements observed with regard to the choroidal metastatic tumour.

Keywords: choroidal neoplasm, choroidal naevus, pulmonary adenocarcinoma, metastases, lung cancer

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Authors: Dwi Arisandi Wijaya, Ernawati Arifin Giri-Rachman, Neni Nurainy

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Typhoid fever disease can be prevented by using a polysaccharide-based vaccine Vi which is a virulence factor of S.typhi. To produce high yield Vi polysaccharide from bacteria, it is important to know the biosynthesis of Vi polysaccharide and the regulators involved. In the In vivo condition, S. typhi faces different osmolarity, and the bacterial two-component system OmpR-EnvZ, regulate by up and down Capsular Vi polysaccharide biosynthesis. A high yielded Vi Polysaccharide strain, S. typhi strain C6524 used to study the effect of media osmolarity on Vi polysaccharide biosynthesis and the osmoregulation pattern of S. typhi strain C6524. The methods were performed by grown S. typhi strain C6524 grown on medium with 50 mM, 100 mM, and 150 mM osmolarity with the batch system. Vi polysaccharide concentration was measured by ELISA method. For further investigation of the osmoregulation pattern of strain C6524, the osmoregulator gene, OmpR, has been isolated and sequenced using the specific primer of the OmpR gene. Nucleotide sequence analysis is done with BLAST and Lallign. Amino Acid sequence analysis is done with Prosite and Multiple Sequence Alignment. The results of cultivation showed the average content of polysaccharide Vi for 50 mM, 100 mM, and 150 mM osmolarities 11.49 μg/mL, 12.06 μg/mL, and 14.53 μg/mL respectively. Analysis using Anova stated that the osmolarity treatment of 150 mM significantly affects Vi content. Analysis of nucleotide sequences shows 100% identity between S. typhi strain C6524 and Ty2. Analysis of amino acid sequences shows that the OmpR response regulator protein of the C6524 strain also has a α4-β5-α5 motif which is important for the regulatory activation system when phosphorylation occurs by domain kinase. This indicates that the regulator osmolarity response of S. typhi strain C6524 has no difference with the response regulator owned by S. typhi strain Ty2. A high Vi response rate in the 150 mM osmolarity treatment requires further research for RcsB-RcsC, another two-component system involved in Vi Biosynthesis.

Keywords: osmoregulator, OmpR, Salmonella, Vi polysaccharide

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Authors: Juliana Ianova, Lyudmila Kabaivanova, Tanya Toshkova- Yotova

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Background: Microalgae are widely known for their nutritional and therapeutic applications due to the richness in nutrients and bioactive elements. The aim of this research was to investigate the growth and production of bioactive compounds with antioxidant properties by different microalgal strains: Scenedesmus acutus M Tomaselli 8, Scenedesmus obliquus BGP, Porphyridium aerugineum and Porphyridium cruentum (Chlorophyta and Rhodophyta). Most of them are freshwater species, with only one marine microalga P. cruentum. Methods: Monoalgal, non-axenic cultures of the investigated strains were grown autotrophically in 200 ml flasks, CO2 - 2% at 132 μmol m-2 s-1 photon flux density and T 25°C. Algal biomass concentration was measured daily by the dry weight. The ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid, C18H18N4O6S4) scavenging assay and CUPRAC assay (cupric ion reducing antioxidant capacity) were used to establish the antioxidant activity of the four algae at the end of the cultivation process, when stationary phase of growth was reached. Results: The highest biomass yield was achieved by Scenedesmus obliquus BGP- (6.6 g/L) after 144 hours of cultivation. Scenedesmus obliquus showed much higher levels of antioxidant properties from the assessed strains. The red microalga Porphyridium aerugineum also exhibits promising reducing antioxidant power. Conclusion: This study confirmed the view that microalgae are promising producers of food supplements and pharmaceuticals.

Keywords: microalgae, dry weight, antioxidant activity, CUPRAC, ABTS

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Authors: Rassoli Aisa, Abrishami Movahhed Arezu, Faturaee Nasser, Seddighi Amir Saeed, Shafigh Mohammad

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Cardiovascular diseases are one of the most common causes of mortality in developed countries. Coronary artery abnormalities and carotid artery stenosis, also known as silent death, are among these diseases. One of the treatment methods for these diseases is to create a deviatory pathway to conduct blood into the heart through a bypass surgery. The saphenous vein is usually used in this surgery to create the deviatory pathway. Unfortunately, a re-surgery will be necessary after some years due to ignoring the disagreement of mechanical properties of graft tissue and/or applied prostheses with those of host tissue. The objective of the present study is to clarify the viscoelastic behavior of human saphenous tissue. The stress relaxation tests in circumferential and longitudinal direction were done in this vein by exerting 20% and 50% strains. Considering the stress relaxation curves obtained from stress relaxation tests and the coefficients of the standard solid model, it was demonstrated that the saphenous vein has a non-linear viscoelastic behavior. Thereafter, the fitting with Fung’s quasilinear viscoelastic (QLV) model was performed based on stress relaxation time curves. Finally, the coefficients of Fung’s QLV model, which models the behavior of saphenous tissue very well, were presented.

Keywords: Viscoelastic behavior, stress relaxation test, uniaxial tensile test, Fung’s quasilinear viscoelastic (QLV) model, strain rate

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Authors: Abdolamir Allameh, Seyyed Mortaza Haghgoo, Adnan Khosravi, Esmaeil Mortaz, Mihan Pourabdollah-Toutkaboni, Sharareh Seifi

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Non-Small Cell Lung Carcinoma (NSCLC) is associated with a number of gene mutations in epidermal growth factor receptor (EGFR). The prognostic significance of mutations in exons 19 and 21, together with serum levels of EGFR, amphiregulin (AR), and Transforming Growth Factor-alpha (TGF-α) are implicated in diagnosis and treatment. The aim of this study was to examine the relationship of EGFR mutations in selected exons with the expression of relevant ligands in sera samples of NSCLC patients. For this, a group of NSCLC patients (n=98) referred to the hospital for lung surgery with a mean age of 59±10.5 were enrolled (M/F: 75/23). Blood specimen was collected from each patient. Besides, formalin fixed paraffin embedded tissues were processed for DNA extraction. Gene mutations in exons 19 and 21 were detected by direct sequencing, following DNA amplification which was done by PCR (Polymerase Chain Reaction). Also, serum levels of EGFR, AR, and TGF-α were measured by ELISA. The results of our study show that EGFR mutations were present in 37% of Iranian NSCLC patients. The most frequently identified mutations were deletions in exon 19 (72.2%) and substitutions in exon 21 (27.8%). The most frequently identified alteration, which is considered as a rare mutation, was the E872K mutation in exon 21, which was found in 90% (9 out of 10) cases. EGFR mutation detected in exon 21 was significantly (P<0.05) correlated with the levels of its ligands, EGFR and TGF-α in serum samples. Furthermore, it was found that increased serum AR (>3pg/ml) and TGF-α (>10.5 pg/ml) were associated with shorter overall survival (P<0.05). The results clearly showed a close relationship between EGFR mutations and serum EGFR and serum TGF-α. Increased serum EGFR was associated with TGF-α and AR and linked to poor prognosis of NSCLC. These findings are implicated in clinical decision-making related to EGFR-Tyrosine kinase inhibitors (TKIs).

Keywords: lung cancer, Iranian patients, epidermal growth factor, mutation, prognosis

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Authors: V. Baweja, K. K. Gupta, V. Dubey, C. Keshavam

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Antimicrobial activity of six indigenous plants such as Tulsi Ocimum sanctum, Neem Azadirachta indica, Aloe vera, Turmeric Curcuma longa, Lantana Lantana camara, and Clove Syzygium aromaticum was assessed against the gut microbiota of the dengue fever mosquito Aedes aegypti, keeping in view that the presence of midgut bacteria may affect the ability of the vector to transmit pathogens. Eleven different types of bacterial clones were isolated from the midgut of lab-reared fourth instar larvae of Aedes aegypti and were grown on LB agar medium at an optimum temperature of 25 ºC. Identification of these bacteria was done on the basis of their colony characteristic such as colony size, shape, opacity, elevation, consistency, and growth. Light microscopic studies of the gut microbiota revealed dominance of Gram-negative cocci over gram positive cocci and bacilli and Gram-negative bacilli. Identification of species was done by chemical characterization of the colonies. Crude extracts of all test plants were screened for their antimicrobial activities against gut microbiota by disc diffusion assay. The zone of exclusion seen after 24 hr of incubation in different assays revealed the most potent antibacterial activities in neem followed by clove and turmeric. Lantana and Aloe vera were least effective.

Keywords: plant extract, aedes, dengue, antimicrobial activity

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Authors: Tunca Gul Altuntas, Aziz Baydar, Cemre Acar, Sezen Yılmaz, Tulay Coban

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Free radicals, which are generated in many bioorganic redox processes, play a role in the pathogenesis of several diseases including cancer, arthritis, hemorrhagic shock, inflammatory, cardiovascular, neurodegenerative diseases and age-related degenerative brain diseases. Exposures of normal cell to free radical damages several structures, oxidizes nucleic acids, proteins, lipids, or DNA. Compounds interfere with the action of reactive oxygen species might be useful in prevention and treatment of these pathologies. A series of indole compounds containing piperazine ring were synthesized. Coupling of indole-2-carboxylic acid with monosubstituted piperazines was accomplished with 1,1’-carbonyldiimidazole (CDI) in a good yield. The structures of prepared compounds were verified in good agreement with their 1H NMR (nuclear magnetic resonance), MS (mass spectrophotometry), and IR (infrared spectrophotometry) characteristics. In this work, all synthetized indole derivatives were screened in vitro for their antioxidative potential against vitamin E (α-tocopherol) using different antioxidant assays such as superoxide anion formation, lipid peroxidation levels in rat liver, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical scavenging activity. The synthesized compounds showed various levels of inhibition compared to vitamin E. This may give promising results for the development of new antioxidant agents.

Keywords: antioxidant, indoles, piperazines, reactive oxygen species

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Authors: Erika K. Méndez, Carlos E. Orrego, Diana L. Manrique, Juan D. Gonzalez, Doménica Vallejo

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High moisture content in fruits generates post-harvest problems such as mechanical, biochemical, microbial and physical losses. Dehydration, which is based on the reduction of water activity of the fruit, is a common option for overcoming such losses. However, regular hot air drying could affect negatively the quality properties of the fruit due to the long residence time at high temperature. Power ultrasound (US) application during the convective drying has been used as a novel method able to enhance drying rate and, consequently, to decrease drying time. In the present study, a new approach was tested to evaluate the effect of US on the drying time, the final antioxidant activity (AA) and the total polyphenol content (TPC) of banana slices (BS), mango slices (MS) and guava slices (GS). There were also studied the drying kinetics with nine different models from which water effective diffusivities (Deff) (with or without shrinkage corrections) were calculated. Compared with the corresponding control tests, US assisted drying for fruit slices showed reductions in drying time between 16.23 and 30.19%, 11.34 and 32.73%, and 19.25 and 47.51% for the MS, BS and GS respectively. Considering shrinkage effects, Deff calculated values ranged from 1.67*10-10 to 3.18*10-10 m2/s, 3.96*10-10 and 5.57*10-10 m2/s and 4.61*10-10 to 8.16*10-10 m2/s for the BS, MS and GS samples respectively. Reductions of TPC and AA (as DPPH) were observed compared with the original content in fresh fruit data in all kinds of drying assays.

Keywords: banana, drying, effective diffusivity, guava, mango, ultrasound

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Authors: Muhammad Munir, Riffat Mehboob, Samina Naeem, Muhammad Salman, Shehryar Ahmed, Irshad Hussain Qureshi, Tahira Murtaza Cheema, Ashraf Sultan, Akmal Laeeq, Nakhshab Choudhry, Asad Aslam Khan, Fridoon Jawad Ahmad

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A dengue outbreak in Lahore, Pakistan during 2011 was unprecedented in terms of severity and magnitude. This research aims to determine the serotype distribution of dengue virus during this outbreak and classify the patients demographically. 5ml of venous blood was drawn aseptically from 166 patients with dengue-like signs to test for the virus between the months of August to November 2011. The samples were sent to the CDC, Atlanta, Georgia for the purpose of molecular assays to determine their serotype. RT-PCR protocol was performed targeting at the 4 dengue serotypes. Out of 166 cases, dengue infection was detected with RT-PCR in 95 cases, all infected with same serotype DEN-2. 75% of positive cases were males while 25% were females. Most positive patients were in the age range of 16-30 years. 33% positive cases had accompanying bleeding. This is first study during the 2011 dengue epidemic in Lahore that reports DEN-2 as the only prevalent serotype. It also indicates that more infected patients were males, adults, within age range of 16-30 years, peaked in the month of November, Dengue hemorrhagic fever (DHF) is manifested more in females, Ravi town was heavily hit by dengue virus infection.

Keywords: dengue, serotypes, Pakistan, DEN 2, Lahore, demography, serotype distrbution, 2011 epidemic

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Authors: Sabir Hussain, Mujtaba Hassan, Kashif Rasool, Asif Shahzad

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Fine particulate matter with an aerodynamic diameter of less than 2.5 μm (PM2.5) was collected in Islamabad from November 2022 to January 2023, at urban sites. The average mass concentrations of PM2.5 varied, ranging from 90.5 to 133 μg m−3 in urban areas. Environmental scanning electron microscopy (ESEM) analysis revealed that Islamabad's PM2.5 comprised soot aggregates, ashes, minerals, bio-particles, and unidentified particles. Results from inductively coupled plasma atomic emission spectroscopy (ICP-OES) indicated a gradual increase in total elemental concentrations in Islamabad PM2.5 in winter, with relatively high levels in December. Significantly different elemental compositions were observed in urban PM2.5. Enrichment factor (EF) analysis suggested that elements such as K, Na, Ca, Mg, Al, Fe, Ba, and Sr were of natural origin, while As, Cu, Zn, Pb, Cd, Mn, Ni, and Se originated from anthropogenic sources. Plasmid DNA assays demonstrated varying levels of potential toxicity in Islamabad PM2.5 collected from urban sites, as well as across different seasons. Notably, the urban winter PM2.5 sample exhibited much stronger toxicity compared to other samples. The presence of heavy metals in Islamabad PM2.5, including Cu, Zn, Pb, Cd, Cr, Mn, and Ni, may have synergistic effects on human health.

Keywords: islamabad particulate matter pm2.5, scanning electron microscopy with energy-dispersive x-ray spectroscopy(sem-eds), fourier transform infrared spectroscopy(ftir), inductively coupled plasma optical emission spectroscopy(icp-oes)

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Authors: Ozlem Oral, Seval Kilic, Ozlem Yedier, Serap Dokmeci, Devrim Gozuacik

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Autophagy is a degradation pathway, activating under stress conditions. It digests macromolecules, such as abnormal proteins and long-lived organelles by engulfing them and by subsequent delivery of the cargo to lysosomes. The members of the phospholipid-dependent serine/threonine kinases, involved in many signaling pathways, which are necessary for the regulation of cellular metabolic activation. Previous studies implicate that, serine/threonine kinases have crucial roles in the mechanism of many diseases depend on the activated and/or inactivated signaling pathway. Data indicates, the signaling pathways activated by serine/threonine kinases are also involved in activation of autophagy mechanism. However, the information about the effect of serine/threonine kinases on autophagy mechanism and the roles of these effects in disease formation is limited. In this study, we investigated the effect of activated serine/threonine kinases on autophagic pathway. We performed a commonly used autophagy technique, GFP-LC3 dot formation and by using microscopy analyses, we evaluated promotion and/or inhibition of autophagy in serine/threonine kinase-overexpressed fibroblasts as well as cancer cells. In addition, we carried out confocal microscopy analyses and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. Based on the shRNA-library based screening, we identified autophagy-related proteins affected by serine/threonine kinases. We further studied the involvement of serine/threonine kinases on the molecular mechanism of newly identified autophagy proteins and found that, autophagic pathway is indirectly controlled by serine/threonine kinases via specific autophagic proteins. Our data indicate the molecular connection between two critical cellular mechanisms, which have important roles in the formation of many disease pathologies, particularly cancer. This project is supported by TUBITAK-1001-Scientific and Technological Research Projects Funding Program, Project No: 114Z836.

Keywords: autophagy, GFP-LC3 dot formation assay, serine/threonine kinases, shRNA-library screening

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Authors: Carolina Pereira Lobato Costa, Cristina Pascual-González, Monica Echeverry, Javier LLorca, Carlos Gonzáléz, Juan Pedro Fernández-Bláquez

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Although the potential of PLA self-reinforced composites for bone applications, not much literature addresses optimal manufacturing conditions. In this regard, this paper describes the woven self-reinforced PLA composites manufacturing processes: the commingling of yarns, weaving, and hot pressing and characterizes the manufactured laminates. Different structures and properties can be achieved by varying the hot compaction process parameters (pressure, holding time, and temperature). The specimens manufactured were characterized in terms of thermal properties (DSC), microstructure (C-scan optical microscope and SEM), strength (tensile test), and biocompatibility (MTT assays). Considering the final device, 155 ℃ for 10 min at 2 MPa act as the more appropriate hot pressing parameters. The laminate produced with these conditions has few voids/porosity, a tensile strength of 30.39 ± 1.21 MPa, and a modulus of 4.09 ± 0.24 GPa. Subsequently to the tensile testing was possible to observe fiber pullout from the fracture surfaces, confirming that this material behaves as a composite. From the results, no single laminate can fulfill all the requirements, being necessary to compromise in function of the priority property. Further investigation is required to improve materials' mechanical performance. Subsequently, process parameters and materials configuration can be adjusted depending on the place and type of implant to suit its function.

Keywords: woven fabric, self-reinforced polymer composite, poly(lactic acid), biodegradable

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Authors: Livy Febria Tedjamulia, Aas Nurasyiah, Ivana Josephin Purnama, Monika Diah Maharani Kusumastuti, Achmad Ridwan Ariyantoro

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Papua's candy is one of Indonesia’s indigenous consumption consisting of areca nut (Areca catechu), forest betel fruit (Piper aduncum), and CaCO3. This research aims to determine the concentration of tannins in areca nut, alkaloids in areca nut, flavonoids in forest betel fruit; detect their interaction and CaCO3; also toform a standardize consumption recommendation. The research methodwas includingDPPH assay for papua’s candy mixture, which resulted in IC50 value. Data analysis used is mathematical linear regression for each experiment. The test result of alkaloid is a Rf value of 0.773, while concentration of tannin and flavonoidare 0.603 mgGAE/g and 125.402 gQE/g, respectively. The IC50 value shows number of 3.0403, showing high antioxidant capacity.Other antioxidant assays were being studied using literature review, namely trolox and oxygen radical absorbance capacity, to figure out interaction among the bioactive compounds. It turned out that the interaction detected is antagonistic, which means the compound that is joined already has a stable molecular structure so that could reduce free radicals by donating hydrogen atoms. The recommendation consumptions given are 4 areca nuts, 5 forest betels, and 1 gram of lime betel. Therefore, papua's candy has its potential to be developed into functional food.

Keywords: antioxidant, bioactive compounds interaction, free radical, papua’s candy

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Authors: Monika Szymańska-Czerwińsk, Kinga Zaręba-Marchewka, Krzysztof Niemczuk

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Chlamydiaceae are Gram-negative bacteria distributed worldwide in animals and humans. One of them is Chlamydia gallinacea recently discovered. Available data show that C. gallinacea is dominant chlamydial agent found in poultry in European and Asian countries. The aim of the studies was screening of poultry flocks in order to evaluate frequency of C. gallinacea shedding and genetic diversity. Sampling was conducted in different regions of Poland in 2019-2020. Overall, 1466 cloacal/oral swabs were collected in duplicate from 146 apparently healthy poultry flocks including chickens, turkeys, ducks, geese and quails. Dry swabs were used for DNA extraction. DNA extracts were screened using a Chlamydiaceae 23S rRNA real-time PCR assay. To identify Chlamydia species, specific real-time PCR assays were performed. Furthermore, selected samples were used for sequencing based on ompA gene fragments and variable domains (VD1-2, VD3-4). In total, 10.3% of the tested flocks were Chlamydiaceae-positive (15/146 farms). The presence of Chlamydiaceae was confirmed mainly in chickens (13/92 farms) but also in turkey (1/19 farms) and goose (1/26 farms) flocks. Eleven flocks were identified as C. gallinacea-positive while four flocks remained unclassified. Phylogenetic analysis revealed at least 16 genetic variants of C. gallinacea. Research showed that Chlamydiaceae occur in a poultry flock in Poland. The strains of C. gallinacea as dominant species show genetic variability.

Keywords: C. gallinacea, emerging agent, poultry, real-time PCR

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Authors: M. Eckert, D. Mendes, J P. Gonçalves, C. Moço, M. Oliveira

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The intensive extraction of natural aggregates leads to both depletion of natural resources and unwanted environmental impacts. On the other hand, uncontrolled disposal of Construction and Demolition Wastes (C&DW) causes the lifetime reduction of landfills. It is known that the European Union produces, each year, about 850 million tons of C&DW. For all the member States of the European Union, one of the milestones to be reached by 2020, according to the Resource Efficiency Roadmap (COM (2011) 571) of the European Commission, is to recycle 70% of the C&DW. In this work, properties of different types of recycled C&DW aggregates and natural aggregates were compared. Assays were performed according to European Standards (EN 13285; EN 13242+A1; EN 12457-4; EN 12620; EN 13139) for the characterization of there: physical, mechanical and chemical properties. Not standardized tests such as water absorption over time, mass stability and post compaction sieve analysis were also carried out. The tested recycled C&DW aggregates were classified according to the requirements of the European Standards regarding there potential use in concrete, mortar, unbound layers of road pavements and embankments. The results of the physical and mechanical properties of recycled C&DW aggregates indicated, in general, lower quality properties when compared to natural aggregates, particularly, for concrete preparation and unbound layers of road pavements. The results of the chemical properties attested that the C&DW aggregates constitute no environmental risk. It was concluded that recycled aggregates produced from C&DW have the potential to be used in many applications.

Keywords: recycled aggregate, sustainability, aggregate properties, European Standard Classification

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Authors: Kina Kim

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Background: There are two types of interferon-gamma release assays (IGRAs) in use for the detection of latent tuberculosis infection (LTBI), QuantiFERON-TB Gold In-tube (QFT-GIT) and T-SPOT.TB. There are some reports that IGRA results are affected by the patient's age. This study aims to compare the results of both IGRA tests according to age groups. Methods: We reviewed 54,882 samples referred to an independent reference laboratory (Seegene Medical Foundation, Seoul, Korea) for the diagnosis of LTBI from January 1, 2021, to December 31, 2021. This retrospective study enrolled 955 patients tested using QFT-GIT and 53,927 patients tested using T-SPOT.TB. The results of both IGRAs were divided in three age groups (0-9, 10-17, and ≥18-year old). The positive rates and the indeterminate rates between QFT-GIT and T-SPOT.TB were compared. We also evaluated the differences in positive and indeterminate rates by age-group. Results: The positive rate of QFT-GIT was 20.1% (192/955) and that of T-SPOT.TB was 8.7% (4704/53927) in overall patients. The positive rates of QFT-GIT in individuals aged 0-9, 10-17, and over 18-year old were 15.4%, 13.3%, and 22.0%, respectively. The positive rates of T-SPOT.TB were 8.9%, 2.0% and 8.8%,in each agegroup, respectively.The overall prevalence of indeterminate results was 2.1% (20/955) of QFT-GIT and 0.5% (270/53927) of T-SPOT.TB. The indeterminate rates of QFT-GIT in individuals aged 0-9, 10-17, and over 18 years were 0.4%, 6.7%, and 2.6%, respectively. The indeterminate rate of T-SPOT.TB were 0.5%, 0.7% and 0.5%,in each age group, respectively. Conclusion: Our findings suggest that T-SPOT.TB has a lower rate of positive results in overall patients and a lower rate of indeterminate results than those of QFT-GIT. The highest positive rate was found in the over 18 years group for QFT-GIT, but the positive rates of T-SPOT.TB was not significantly different among groups by age. QFT-GIT showed variable and higher indeterminate rates according to age group, but T-SPOT.TB showed lower rates in all age groups(<1%).

Keywords: LTBI, IGRA, QFT-GIT, T-SPOT. TB

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Authors: Fadilah Aleanizy

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Colicins are a family of bacterial toxins that kill Escherichia coli and other closely related species. The mode of action of colicins involves binding to an outer membrane receptor and translocation across the cell envelope, leading to cytotoxicity through specific targets. The mechanism of colicin cytotoxicity includes a non-specific endonuclease activity or depolarization of the cytoplasmic membrane by pore-forming activity. For Group A colicins, translocation requires an interaction between the N-terminal domain of the colicin and a series of membrane- bound and periplasmic proteins known as the Tol system (TolB, TolR, TolA, TolQ, and Pal and the active domain must be translocated through the outer membranes. Protein-protein interactions are intrinsic to virtually every cellular process. The transient protein-protein interactions of the colicin include the interaction with much more complicated assemblies during colicin translocation across the cellular membrane to its target. The potassium release assay detects variation in the K+ content of bacterial cells (K+in). This assays is used to measure the effect of pore-forming colicins such as ColA on an indicator organism by measuring the changes of the K+ concentration in the external medium (K+out ) that are caused by cell killing with a K+ selective electrode. One of the goals of this work is to employ a quantifiable in-vivo method to spot which Tol protein are more implicated in the interaction with colicin A as it is translocated to its target.

Keywords: K+ efflux, Colicin A, Tol-proteins, E. coli

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Authors: Ana P. R. N. Barroso, Maurício N. Kleinberg, Frederico R. Silva, Rodrigo F. Guimarães, Marcelo M. V. Parente, Walney S. Araújo

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The fight against accelerated wear of the equipment used in the oil and gas sector is a challenge for minimizing maintenance costs. Corrosion being one of the main agents of equipment deterioration, we seek alternative materials that exhibit improved corrosion resistance at low cost of production. This study aims to evaluate the corrosion behavior of experimental alloys containing 15% and 17% of chromium (Cr) and 5% of molybdenum (Mo) in comparison with an AISI 444 commercial alloy. Microstructural analyzes were performed on samples of the alloys before and after the electrochemical tests. Two samples of each solubilized alloy were also taken for analysis of the corrosion behavior by testing potentiodynamic polarization (PP) and Electrochemical Impedance Spectroscopy (EIS) with immersion time of 24 hours in electrolytic solution with acidic character. The graphics obtained through electrochemical tests of PP and EIS indicated that among the experimental alloys, the alloy with higher chromium content (17%) had a higher corrosion resistance, confirming the beneficial effect of adding chromium. When comparing the experimental alloys with the AISI 444 commercial alloy, it is observed that the AISI 444 commercial alloy showed superior corrosion resistance to that of the experimental alloys for both assays, PP and EIS. The microstructural analyzes performed after the PP and EIS tests confirmed the results previously described. These results suggest that the addition of these levels of molybdenum did not favor the electrochemical behavior of experimental ferritic alloys for the electrolytic medium studied.

Keywords: corrosion, molybdenum, electrochemical tests, experimental alloys

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Authors: J. Švarc-Gajić, A. Cvetanović

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Apigenin is a flavone synthetized by many plants and quite abundant in chamomile (Matricaria recutita) in its free form and in the form of its glucoside and different acylated forms. Many beneficial health effects have been attributed to apigenin, such as chemo-preventive, anxiolytic, anti-inflammatory, antioxidant and antispasmodic. It is reported that free apigenin is much more bioactive in comparison to its bound forms. Subcritical water offers numerous advantages in comparison to conventional extraction techniques, such as good selectivity, low price and safety. Superheated water exhibits high hydrolytical potential which must be carefully balanced when using this solvent for the extraction of bioactive molecules. Moderate hydrolytical potential can be exploited to liberate apigenin from its bound forms, thus increasing biological potential of obtained extracts. The polarity of pressurized water and its hydrolytical potential are highly dependent on the temperature. In this research chamomile ligulate flowers were extracted by pressurized hot water in home-made subcritical water extractor in conditions of convective mass transfer. The influence of the extraction temperature was investigated at 30 bars. Extraction yields of total phenols, total flavonoids and apigenin depending on the operational temperature were calculated based on spectrometric assays. Optimal extraction temperature for maximum yields of total phenols and flavonoids showed to be 160°C, whereas apigenin yield was the highest at 120°C.

Keywords: superheated water, temperature, chamomile, apigenin

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Authors: Alok Raghav, Jamal Ahmad

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Background: Serum albumin glycation and advanced glycation end products (AGE) formation correlates in diabetes and its associated complications. Extensive modified human serum albumin is used to study the biochemical, electrochemical and functional properties in hyperglycemic environment with relevance to diabetes. We evaluate Spectroscopic, side chain modifications, amino acid analysis, biochemical and functional group properties in four glucose modified samples. Methods: A series four human serum albumin samples modified with glucose was characterized in terms of amino acid analysis, spectroscopic properties and side chain modifications. The diagnostic technique employed incorporates UV Spectroscopy, Fluorescence Spectroscopy, biochemical assays for side chain modifications, amino acid estimations, electrochemical and optical characterstic of glycated albumin. Conclusion: Glucose modified human serum albumin confers AGEs formation alters biochemical, electrochemical, optical, and functional property that depend on the reactivity of glucose and its concentration used for in-vitro glycation. A biochemical, electrochemical, optical, and functional characterization of modified albumin in-vitro produced AGE product that will be useful to interpret the complications and pathophysiological significance in diabetes.

Keywords: human serum albumin, glycated albumin, adavanced glycation end products, associated pathologies

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Authors: Rafael Estrada, Cristian Ruiz Rueda

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Carbapenems are last-resort antibiotics used in clinical settings to treat antibiotic-resistant bacterial infections. Thus, the emergence and spread of resistance to carbapenems is a major public health concern. Here, we have studied a carbapenem-resistant Aeromonas veronii strain previously isolated from a water sample from Sam Simeon Creek (Hearst San Simeon State Park, CA). Analysis of this isolate using disk-diffusion, CarbaNP, eCIM and mCIM assays revealed that it was resistant to amoxicillin-clavulanic acid and all carbapenems tested and that this isolate produced a potentially novel carbapenemase of the Metallo-β-lactamase family. Whole genome sequencing analysis revealed that this A. veronii isolate carries a novel variant of the blacₚₕₐ class β-carbapenemase gene that was closely related to the blacₚₕₐ₇ gene of Aeromonas jandaei. This isolate also carried a novel variant of the blaₒₓₐ class D carbapenemase gene that was most closely related to the blaₒₓₐ-₉₁₂ gene found in other Aeromonas veronii isolates. Finally, we also identified a novel class C β-lactamase gene moderately related to the blaFₒₓ-₁₇ gene of Providencia stuartii and other blaFₒₓ variants identified in Klebsiella pneumoniae, Escherichia coli and other Enterobacteriaceae. Overall, our findings reveal that environmental isolates are an important reservoir of multiple carbapenemases and other β-lactamases of clinical significance.

Keywords: β-lactamases, carbapenem, antibiotic-resistant, aeromonas veronii

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Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

Abstract:

One of the major interferences in the Philippines’ tuberculosis control program is the widespread prevalence of Mtb strains that are resistant to known drugs, such as the MDR-TB (Multi Drug Resistant Tuberculosis) and XDR-TB (Extensively Drug Resistant Tuberculosis). Therefore, there is a pressing need to search for novel Mtb drug targets in order to be able to combat these drug resistant strains. The enzyme 7,8-diaminopelargonic acid aminotransferase enzyme, or more commonly known as BioA, is one such ideal target, as it is known that humans do not possess this enzyme. BioA primarily plays a key role in Mtb’s lipid biosynthesis pathway; more specifically in the synthesis of the enzyme cofactor biotin. In this study, structure-based pharmacophore screening, docking, and ADMET evaluation of compounds obtained from the DrugBank chemical database were performed against the MtbBioA enzyme. Results of the screening, docking, ADMET, and TOPKAT calculations revealed that out of the 6,516 compounds in the library, only 7 compounds indicated more favorable binding energies as compared to the enzyme’s known inhibitor, amiclenomycin (ACM), as well as good solubility and toxicity properties. Moreover, out of these 7 compounds, Molecule 6 exhibited the best solubility and toxicity properties. In the future, these lead compounds may then be subjected to bioactivity assays in vitro or in vivo for further evaluation of its therapeutic efficacy.

Keywords: 7, 8-diaminopelargonic acid aminotransferase, BioA, pharmacophore, molecular docking, ADMET, TOPKAT

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Authors: Roman Khanferyan

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Endurance exercises and strenuous muscle activity are accompanied by multiple immune dysfunctions due to the activation of cytokines and chemokines synthesis. This study assesses changes in the synthesis of immune regulatory mediators in elite athletes during endurance sports activity. The concentrations of cytokines/chemokines (IL-2, IL-6, IL-8, IL-10, IL-18, MIP-1 beta, GRO-alpha, RANTES, SDF-1a, VEGF) in sera of hockey athletes (n=33) and in supernatants of 24-h cultivated peripheral blood mononuclear cells (PBMC) of boxers (n=6) assayed by ELISA and Luminex xMAP multiplex assays. Estimation of body composition studied by using bioimpedance technology. The dietary energy consumption per person has been estimated using an album of different sizes of portions of the most frequently consumed foods. It has been demonstrated that endurance sports activity enhances the secretions of most pro- and anti-inflammatory cytokines and chemokines in more than 2-6 fold. The study demonstrated that the high increase of more than 3-4 times in the concentration of IL-18 in sera of athletes (327.86 + 45.67 pg/ml) didn’t correlate with BMI (p=0.040) but demonstrated a low correlation with MMI (p=0.234) and BMR (p=0,231). The opposite impact on the concentration of IL-10 has been demonstrated in athletes. It has been shown a negative correlation between its concentration and BMI (p= - 0.251), MMI (p= - 0.327), and BMR (p= - 0.301). In vitro studies in boxers showed greater amounts of chemokines in the PBMC supernatants, including MIP-1β, GRO-α, RANTES, SDF-1α, and IL-8 (P<0.05). At the same time, healthy controls had greater supernatant levels of MCP-1, Eotaxin, and MIP-1α. The study demonstrated a high correlation between physical activity, usual athletes' diet, and consumption of specialized sports nutrition products.

Keywords: sport nutrition, cytokines, chemokines, endurace exercises

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Authors: Syed Dawood Md. Taimur, Md. Hasanur Rahman, Syeda Fahmida Afrin, Farzana Islam

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The concept of myocardial injury, although first recognized from animal studies, is now recognized as a clinical phenomenon that may result in microvascular damage, no-reflow phenomenon, myocardial stunning, myocardial hibernation and ischemic preconditioning. The final consequence of this event is left ventricular (LV) systolic dysfunction leading to increased morbidity and mortality. The typical clinical case of reperfusion injury occurs in acute myocardial infarction (MI) with ST segment elevation in which an occlusion of a major epicardial coronary artery is followed by recanalization of the artery. This may occur either spontaneously or by means of thrombolysis and/or by primary percutaneous coronary intervention (PCI) with efficient platelet inhibition by aspirin (acetylsalicylic acid), clopidogrel and glycoprotein IIb/IIIa inhibitors. In recent years, percutaneous coronary intervention (PCI) has become a well-established technique for the treatment of coronary artery disease. PCI improves symptoms in patients with coronary artery disease and it has been increasing the safety of procedures. However, peri- and post-procedural myocardial injury, including angiographical slow coronary flow, microvascular embolization, and elevated levels of cardiac enzyme, such as creatine kinase and troponin-T and -I, has also been reported even in elective cases. Furthermore, myocardial reperfusion injury at the beginning of myocardial reperfusion, which causes tissue damage and cardiac dysfunction, may occur in cases of the acute coronary syndrome. Because patients with myocardial injury is related to larger myocardial infarction and have a worse long-term prognosis than those without myocardial injury, it is important to prevent myocardial injury during and/or after PCI in patients with coronary artery disease. To date, many studies have demonstrated that adjunctive pharmacological treatment suppresses myocardial injury and increases coronary blood flow during PCI procedures. In this review, we highlight the usefulness of pharmacological treatment in combination with PCI in attenuating myocardial injury in patients with coronary artery disease.

Keywords: coronary artery disease, percutaneous coronary intervention, myocardial injury, pharmacology

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Authors: Yong-Hui Zheng

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Viruses hijack host machineries to propagate and spread, which disrupts cellular homeostasis and activates various counteractive mechanisms. Infection of enveloped viruses is dependent on their fusion proteins, which bind to viral receptors to allow virus entry into cells. Fusion proteins are glycoproteins and expressed in the endoplasmic reticulum (ER) by hijacking the secretory pathway. Previously, we reported that Zaire ebolavirus (EBOV)-glycoprotein (GP) expression induces ER stress, and EBOV-GP is targeted by the calnexin cycle to macro-ER-phagy for degradation. We now report that expression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/SARS2)-spike (S) protein also causes ER stress, and its expression is strongly downregulated by mannosidase alpha class 1B member 1 (MAN1B1), a class I α-mannosidase from the ER. MAN1B1 co-localizes with SARS2-S in the ER, and its downregulation of SARS2-S is blocked by inhibitors targeting lysosomes and autophagy, but not proteasomes, indicating SARS2-S degradation by autolysosomes. Notably, the SARS2-S degradation does not require the core autophagy machinery including ATG3, ATG5, ATG7, and phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3)/vacuolar protein sorting 34 (VPS34), and instead, it requires Beclin 1 (BECN1), a core component in the PI3KC3 complex. In addition, MAN1B1 does not trigger SARS2-S polyubiquitination, and consistently, the SARS2-S degradation does not require the autophagy receptor sequestosome 1 (SQSTM1)/p62. MAN1B1 also downregulates EBOV-GP similarly, but this degradation does not require BECN1. Collectively, we conclude that MAN1B1 downregulates viral fusions by micro-ER-phagy, and importantly, we have identified BECN1-dependent and BECN1-independent mechanisms for micro-ER-phagy.

Keywords: Micro-ER-phagy, reticulophagy, fusion proteins, ER stress

Procedia PDF Downloads 66

Authors: İ. Çelik, Goksel Demirer

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Turkey is the third largest producer of pistachio (Pistacia vera L.) after Iran and United States. Harvested pistachio nuts are covered with organic hull which is removed by de-hulling process. Most of the pistachio by-products which are produced during de-hulling process are considered as agricultural waste and often mixed with soil, to a lesser extent are used as feedstuff by local livestock farmers and a small portion is used as herbal medicine. Due to its high organic and phenolic content as well as high solids concentration, pistachio processing wastes create significant waste management problems unless they are properly managed. However, there is not a well-established waste management method compensating the waste generated during the processing of pistachios. This study investigated the anaerobic treatability and biogas generation potential of pistachio hull waste. The effect of pre-treatment on biogas generation potential was investigated. For this purpose, Biochemical Methane Potential (BMP) Assays were conducted for two Chemical Oxygen Demand (COD) concentrations of 22 and 33 g tCOD l-1 at the absence and presence of chemical and thermal pre-treatment methods. The results revealed anaerobic digestion of the pistachio de-hulling wastes and subsequent biogas production as a renewable energy source are possible. The observed percent COD removal and methane yield values of the pre-treated pistachio de-hulling waste samples were significantly higher than the raw pistachio de-hulling waste. The highest methane yield was observed as 213.4 ml CH4/g COD.

Keywords: pistachio de-hulling waste, biogas, renewable energy, pre-treatment

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Authors: Wanwisa Deenin, Abdulhadee Yakoh, Chahya Kreangkaiwal, Orawon Chailapakul, Kanitha Patarakul, Sudkate Chaiyo

Abstract:

LipL32 is an outer membrane protein present only on pathogenic Leptospira species, which are the causative agent of leptospirosis. Leptospirosis symptoms are often misdiagnosed with other febrile illnesses as the clinical manifestations are non-specific. Therefore, an accurate diagnostic tool for leptospirosis is indeed critical for proper and prompt treatment. Typical diagnosis via serological assays is generally performed to assess the antibodies produced against Leptospira. However, their delayed antibody response and complicated procedure are undoubtedly limited the practical utilization especially in primary care setting. Here, we demonstrate for the first time an early-stage detection of LipL32 by an integrated lateral-flow immunoassay with electrochemical readout (eLFIA). A ferrocene trace tag was monitored via differential pulse voltammetry operated on a smartphone-based device, thus allowing for on-field testing. Superior performance in terms of the lowest detectable limit of detection (LOD) of 8.53 pg/mL and broad linear dynamic range (5 orders of magnitude) among other sensors available thus far was established. Additionally, the developed test strip provided a straightforward yet sensitive approach for diagnosis of leptospirosis using the collected human sera from patients, in which the results were comparable to the real-time polymerase chain reaction technique.

Keywords: leptospirosis, electrochemical detection, lateral flow immunosensor, point-of-care testing, early-stage detection

Procedia PDF Downloads 88

Authors: Berredjem Hajira, Benlaifa Meriem, Becheker Imene, Bardi Rafika, Djebar Med Reda

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Recent acquired or reactivation T.gondii infection is a serious complication in HIV patients. Classical serological diagnosis relies on the detection of anti-Toxoplasma immunoglobulin ; however, serology may be unreliable in HIV immunodeficient patients who fail to produce significant titers of specific antibodies. PCR assays allow a rapid diagnosis of Toxoplasma infection. In this study, we compared the value of the PCR for diagnosing active toxoplasmosis in cerebrospinal fluid and blood samples from HIV patients. Anti-Toxoplasma antibodies IgG and IgM titers were determined by ELISA. In parallel, nested PCR targeting B1 gene and conventional PCR-ELISA targeting P30 gene were used to detect T. gondii DNA in 25 blood samples and 12 cerebrospinal fluid samples from patients in whom toxoplasmic encephalitis was confirmed by clinical investigations. A total of 15 negative controls were used. Serology did not contribute to confirm toxoplasmic infection, as IgG and IgM titers decreased early. Only 8 out 25 blood samples and 5 out 12 cerebrospinal fluid samples PCRs yielded a positive result. 5 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples. However, conventional nested B1 PCR gave best results than the P30 gene one for the detection of T.gondii DNA in both samples. All samples from control patients were negative. This study demonstrates the unusefulness of the serological tests and the high sensitivity and specificity of PCR in the diagnosis of toxoplasmic encephalitis in HIV patients.

Keywords: cerebrospinal fluid, HIV, Toxoplasmosis, PCR

Procedia PDF Downloads 373